Streptomycin solution

Primary BMDM cell cultures were established from bone marrow monocytes isolated from 6 Tent5a^Flox/Flox/Tent5c^−/− and 6 wild-type mice. Young adult animals of both sexes (12–25 weeks old) were sacrificed by cervical dislocation, after which femurs and tibias were isolated, and bone marrow was harvested using a centrifugation-based protocol. Material isolated from multiple individuals (siblings of the same sex) was mixed to obtain number of cells sufficient for subsequent analyses. Bone marrow cells were then plated in IMDM medium (Thermo Fisher Scientific; cat. no: 21980065) supplemented with 10% FBS (Gibco), 100 U/ml penicillin/0.1 mg/ml streptomycin solution (Sigma-Aldrich), and 10 ng/ml macrophage colony-stimulating factor (M-CSF, Preprotech; cat. no: 315-02) and cultured at 37 °C in 5% CO₂. On the day before the vaccine treatment, 0.5–1 × 10⁶ cells were seeded on a 6-well plate in the abovementioned medium. The 1 µl of Moderna mRNA-1273 in original LNPs formulation was diluted in 150 µl of Opti MEM medium (Thermo Fisher Scientific) at RT. After 10 min the mixture was added dropwise to the cells and gently mixed. Cells were harvested at 0, 4, 12, 24, 48 and 72 h time points. Total RNA was isolated from cells with TRIzol (Thermo Fisher Scientific), according to the manufacturer’s instructions. 3.5–5 µg of total mRNA was mixed with 50–200 ng oligo-(dT)25-enriched mRNA from S. cerevisiae and standards with predefined poly(A) lengths, followed by library preparation.

The murine colorectal carcinoma MC-38 cells are syngeneic to the C57BL/6 strain. Their origins and metastatic properties have been described in detail previously (12 (link)). MC-38 cells were originally from an NCI repository and were obtained as a kind gift from Dr. Shoshana Yakar (New York University, NY). They were authenticated by Didion and colleagues using single-nucleotide polymorphism profiling (13 (link)). The murine pancreatic ductal adenocarcinoma (PDAC) line KPC FC1199, referred to here as FC1199, was generated in the Tuveson laboratory (Cold Spring Harbor Laboratory, New York, USA) from a PDAC tumor that arose in the genetically engineered Kras G12D/+; p53R172H/+; Pdx1 Cre (KPC) mouse of a pure C57BL/6 background, as described elsewhere (14 (link)) and were a kind gift from Dr. Andrew Lowy (Moores Cancer Center, La Jolla, CA). The cells were tested for common murine pathogens and for mycoplasma contamination (last mycoplasma test performed in August 2021 using a Mycoplasma PCR detection Kit – Abcam), as per the McGill University Animal Care Committee and the McGill University Biohazard Committee guidelines. The cells were maintained as a frozen stock and cultured in vitro for up to 4 weeks only, prior to use in the in vivo experiments, to minimize contamination, genetic drifts, and changes to their metastatic phenotypes. They were cultured in DMEM medium (Wisent), supplemented with 4 mmol/L L-glutamine, 4.5 g/L glucose, 100 U/mL penicillin, and 100 μg/mL streptomycin solution (Sigma-Aldrich, Burlington, Massachusetts, USA), also containing 2 g/L sodium pyruvate and 10% fetal bovine serum (FBS; Wisent) and incubated at 37°C in a humidified incubator with 5% CO₂.

Embrionic zebrafish fibroblast at 1dpf PAC2 cells were cultured under standard conditions (28°C; room atmosphere) in Leibovit's L-15 medium (Sigma-Aldrich Co, St. Luis, MO, US) supplemented with 40% of heat-inactivated fetal bovine serum (Lonza Basel, Switzerland), 20 mM penicillin, erythromycin, streptomycin solution (Sigma-Aldrich Co), 20 mM glutamax (Invitrogen Co), 50 mg Gentamicine sulfate salt (Sigma-Aldrich Co). Human neuroblastoma SH-SY5Y cells were cultured under standard conditions (37°C; 95% air:5% CO2) in 50% MEM and 50% F12 nutrient medium (Sigma-Aldrich Co), supplemented with 10% fetal bovine serum, 2 mM glutamax, and 1% of a penicillin-streptomycin solution. pcDNA3.1Zeo- plasmid transfections were performed by Lipofectamine 2000 (Invitrogen Co) following manufacturer's protocol.

Total RNA from human tissues, the brain, kidney, pancreas, liver, and small intestine was purchased from Clontech (Clontech‐Takara Bio). Five micrograms of total RNA was reverse transcribed using oligo(dT) primers and the SuperScript III First‐Strand Synthesis System (Thermo Fisher Scientific). Full‐length cDNAs of AQPs were isolated from the kidney (AQP1, 2, 3, 7), brain, (AQP4), lung (AQP5), pancreas (AQP8), liver (AQP9), and small intestine (AQP10) by RT‐PCR using primers designed based on the genomic database (Table 1). cDNAs were subcloned into pGEMHE (Liman et al., 1992 (link)) and sequenced to confirm the absence of PCR errors.
Xenopus laevis oocytes were dissociated with collagenase as described previously (Romero et al., 1998 (link)) and injected with either 50 nl of water (control) or a solution containing cRNA at 0.5 ng/nl (25 ng/oocyte), using a Nanoject II injector (Drummond Scientific). Oocytes were incubated at 16°C in OR3 medium and studied for 3–4 days after injection. One liter of OR3 medium contained 0.7% w/v powdered Leibovitz L‐15 medium with L‐Glutamine (Thermo Fisher Scientific), 50 ml of 10,000 U penicillin and 10,000 U streptomycin solution in 0.9% NaCl (Sigma‐Aldrich), and 5 mM HEPES, pH 7.50, and the osmolarity was adjusted to 200 mosmol/kg with NaCl powder (Romero et al., 1998 ). All Xenopus care and oocyte harvest protocols were in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.” Frogs were housed and cared for in accordance with the approval of the Institutional Animal Care and Use Committee of the Mayo Clinic College of Medicine, and in accordance with a manual approved by the Institutional Animal Experiment Committee of the Tokyo Institute of Technology.
To analyze the effects of cRNA amounts injected into oocytes on their activity, oocytes were injected with either 50 nl of water (control) or a solution containing AQP9 cRNA at 0.5 ng/nl (25 ng/oocyte), 0.2 ng/nl (10 ng/oocyte), 0.08 ng/nl (4 ng/oocyte), 0.032 ng/nl (1.6 ng/oocyte), or 0.013 ng/nl (0.64 ng/oocyte), incubated at 16 °C in OR3 medium, and studied for 4 days after injection.